Methods for treatment of inflammatory and infectious viral diseases

ABSTRACT

Methods and therapeutic treatments of diseases such as viral infections are provided including applying peg-Arginase I. Methods are provided that treat inflammation mediated diseases with peg-Arginase I.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure is a continuation-in-part of U.S. patentapplication Ser. No. 13/828,669, now issued as U.S. Pat. No. 8,877,183and entitled “Methods for Treatment of Ocular Diseases” filed on Mar.14, 2013, which application claims benefit of U.S. ProvisionalApplication 61/664,464 filed Jun. 26, 2012, all of which areincorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants P20RR021970and P20GM103501 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

The present disclosure relates generally to treatment of diseases. Moreparticularly, in exemplary though non-limiting embodiments, the presentdisclosure relates to therapeutic methods to treat viral infections andinflammation mediated diseases.

BACKGROUND

Viral diseases are caused by a combination of virus-mediated cytopathiceffects and an acutely overactive (cytokine storm) or chronicinflammatory response. The immune response, intended to control theinfection, frequently exacerbates the disease and causes complications,such as blindness (viral stromal keratitis), encephalitis (West Nilevirus infections), acute respiratory distress syndrome (pandemicinfluenza) and even death. Effective anti-viral drugs in general do notprevent inflammatory complications and therefore, corticosteroids arefrequently required. However, steroids can exacerbate viral replicationresulting in a difficult to interrupt vicious cycle where viralreplication and inflammation cannot be controlled at the same time andin the end result in severe damage of the tissues. Accordingly, there isneed for improved therapies that may be able to control viralreplication and inflammation simultaneously and therefore prevent damageto the tissues.

Improved therapies to control viral replication and information havepotential application in severe viral infections of the eye. The corneais the highly transparent outer-most layer of the eye that provides alarge portion of the eye's refractive power and shields againstinfection by pathogens. Since even the smallest of cells can affectvisual acuity, normal corneal tissue lacks all vasculature and isimmunologically privileged to reduce influx of inflammatory cells.Generally, in the absence of ocular disease and/or trauma, the lack ofvasculature and inflammatory response of the cornea ensures visualacuity is maintained. However, under certain ocular situations, the lackof vascularization and/or suppressed inflammatory response may becompromised.

For example, trauma to the eye by a foreign object (e.g. dust, sand, ormechanical), a surgical procedure (e.g. refractive surgery ortransplantation), or infection of the cornea with viruses, bacteria, orother infectious agents, may initiate an inflammatory reaction mediatedby the immune response and neovascularization (formation of new bloodvessels). These processes may result in destructive cytopathic causesand may lead to thickening and opacification (clouding) of the cornea,which may ultimately lead to a reduction of visual clarity and sometimessight.

Trauma of the cornea (e.g., surgical or accidental) may lead to pain,inflammation, scarring, neovascularization, and/or discharge. Even inthe absence of a particular infectious agent, corneal lesions can affectvisual acuity and progress to further damage to the eye. Generally,corneal trauma may be typically treated with strong corticosteroidswhich may decrease inflammation. However, these agents may have severesecondary effects, including: 1) increased risk of infections (common inocular trauma) by diminishing the protective immune response; 2) delayedhealing of the cornea; 3) loss of an intact epithelial barrier; 4)increased ocular pressure; and 5) eventual deterioration of vision.

Globally, infection- and inflammation-associated eye diseases are theleading causes of corneal blindness and visual morbidity, with over 500million individuals affected. Pathogen-associated ocular diseases are acomplex combination of pathogen-mediated trauma and host-mediatedpathologies, often with the most severe sequelae being a consequence ofhost inflammatory responses. Corneal infections are often treated with acombination of antimicrobials to eliminate pathogens and corticosteroidsto reduce inflammation. However, several problems may arise from thisapproach.

First, effective antimicrobials are not always available for theinfectious agents of the eye. For example, Adenoviral infections, whichmay cause epidemic outbreaks of highly contagious keratoconjunctivitis(pink eye) that can last up to a month, are not readily treatable byantimicrobials. Second, even when anti-viral medications are availableand can kill the viruses, they cannot control the inflammatory responsetriggered by the infection. Typically, inflammation accounts for theclinical presentation of an ocular disease. Often the inflammatoryresponse may cause secondary but more severe damage of the cornea.Third, many pathogens evolve drug resistance, which enables the pathogento replicate even in the presence of the antimicrobial. For example,some strains of herpes simplex viruses (HSV), which are the leadingcause of infectious corneal blindness in the United States, andChlamydia trachomatis, a leading cause of infectious blindnessworldwide, have developed drug resistant strains. Fourth,co-administration of steroids to suppress host anti-pathogen immuneresponses (inflammation) in infected individuals also blocks theprotective role of the immune responses and enables uncontrolledreplication, as well as pathogen-mediated disease progression.

Four herpes viruses are linked to severe infections of the eye that canresult in blindness: HSV-1, HSV-2, VZV, and CMV. The National EyeInstitute estimates that 450,000 Americans have experienced some form ofocular herpetic disease, with 50,000 new and recurrent cases diagnosedannually. Despite effective antivirals against HSV, approximately 25% ofthese cases develop serious inflammation-associated stromal keratitis.Individuals that have experienced ocular herpes have a 50% chance ofrecurrence with each repeated episode triggering deleterious CD4 and CD8T cell responses that can result in scarring of the cornea and aneventual need for corneal transplantation. Although cornealtransplantation restores the patient's sight, it does not cure thepatient of his or her lifelong herpetic infection; therefore,recrudescence of infection in these individuals may renew the viciouscycle and result in damage to the implanted cornea.

Herpetic stromal keratitis (HSK), a blinding eye disease associated withHSV-1 infection, is not simply a virus-mediated disease of the cornealstroma, but a virus-associated chronic immuno-inflammatory disease ofthe eye. It is mediated by many complex immune mechanisms includingmacrophages, dendritic cells, T cells (Th1, Th2, T-regs and Th17),antibodies and even cytokines. Therefore, even with anti-herpetic drugscontrolling HSV-1 replication, the vision threatening disease progressesthrough immune-mediated pathologies.

Clinical presentations of ocular diseases are a complex combination oftrauma and host-mediated inflammation-associated pathologies that mustbe simultaneously controlled and resolved to prevent development ofvision-threatening sequelae. Currently, no treatments exist that caneffectively remedy all of these issues within the eye.

SUMMARY

In an embodiment of the present invention, a method of simultaneouslyinhibiting inflammation and neovascularization of corneal tissue of aneye and promoting healing of the eye is provided, including applyingpeg-Arginase I to the eye. The peg-Arginase I is applied exogenously.The eye may have been infected with an infectious agent. The infectiousagent may be a virus. The peg-Arginase I may inhibit replication of thevirus. The infectious agent may be a bacterium.

The eye may have been traumatized. The trauma may be from a medicalprocedure. The trauma may be from an accidental injury of the eye.

The peg-Arginase I may part of an ophthalmic formulation. The ophthalmicformulation may include peg-Arginase I and free peg molecules. Theophthalmic formulation may further include pH buffers. The ophthalmicformulation may be an eye drop. The eye may be a mammalian eye. Themammalian eye may be a human eye.

According to an embodiment of the present invention, a therapeuticmethod to treat an ocular disease is provided, including applyingpeg-Arginase I to an affected eye. The peg-Arginase I is part of anophthalmic formulation. The affected eye may present at least one ofinflammation and neovascularization. The ocular disease may be caused byan infectious agent. The infectious agent may be a virus. Thepeg-Arginase I may inhibit replication of the virus. The infectiousagent may be a bacterium.

The ocular disease may be trauma to the affected eye. The trauma may befrom a medical procedure. The ocular disease may be a corneal lesion.

According to an embodiment of the present invention, an anti-viraltreatment is provided, including applying peg-Arginase I to a patientinfected by a virus. The treatment may include administering free pegmolecules with the peg-Arginase I to the patient. The peg-Arginase I maybe part of an ophthalmic formulation. The ophthalmic formulation mayinclude peg-Arginase I and free peg molecules. The peg-Arginase may bepart of a respiratory inhalant formulation. The respiratory inhalantformulation may include peg-Arginase I and free peg molecules.Replication of the virus may be inhibited. The patient may be infectedby at least one of HSV-1, HSV-2, CMV, VZV, Adenovirus and Influenzavirus. The at least one of HSV-1, HSV-2, CMV, VZV, Adenovirus andInfluenza virus may be drug resistant. The virus may have infected arespiratory system of the patient. The virus may have infected an ocularregion of the patient.

According to an exemplary embodiment of the present invention, a methodto promote healing of corneal trauma is provided, including applyingpeg-Arginase I to an affected eye. The trauma may be induced by at leastone of accidental injury, surgical procedure, mechanical impact, andpathogenic infection.

According to an exemplary embodiment of the present invention, a methodto treat ocular lesions is provided including applying peg-Arginase I toan affected eye. The peg-Arginase I is part of an ophthalmicformulation. The ophthalmic formulation may be comprised of peg-ArginaseI and free peg molecules.

According to an exemplary embodiment of the present invention, a methodto prevent ocular neovascularization is provided, including applyingpeg-Arginase I to an affected eye. The peg-Arginase I is part of anophthalmic formulation. The ophthalmic formulation may be comprised ofpeg-Arginase I and free peg molecules.

According to an exemplary embodiment of the present invention, a methodto prevent ocular inflammation is provided, including applyingpeg-Arginase I to an affected eye. The peg-Arginase I is part of anophthalmic formulation. The ophthalmic formulation may be comprised ofpeg-Arginase I and free peg molecules.

DESCRIPTION OF DRAWINGS

FIG. 1A is a series of images of rabbit eyes with an 8 mm (area=200 mm²)region of cornea surgically debrided and treated with peg vehicle orpeg-Arginase I, according to an example embodiment of the presentinvention.

FIG. 1B is a line graph showing areas of the corneal lesions over aperiod of six days, as represented by eyes in FIG. 1A.

FIG. 2 is a line graph showing combined ocular clinical scores over aperiod of six days, as represented by eyes in FIG. 1A.

FIG. 3 is a line graph showing corneal neovascularization over a periodof six days, as represented by eyes in FIG. 1A.

FIG. 4 is a line graph showing scleral inflammation over a period of sixdays, as represented by eyes in FIG. 1A.

FIG. 5 is a bar graph showing infectious viral yield for cell culturesinfected with HSV-1 and treated with a vehicle (peg vehicle),peg-Arginase I, or different concentrations of acyclovir, according toan example embodiment of the present invention.

FIG. 6 is a series of images showing virus-mediated cytopathic effectson cell cultures treated as described in FIG. 5.

FIG. 7A is a series of images of HSV-plaques showing capacity forcell-to-cell spread following treatment with a vehicle (peg vehicle),peg-Arginase I, or acyclovir, according to an example embodiment of thepresent invention.

FIG. 7B is a bar graph showing effects of treatment on cell-to-cellspread of HSV-1 for cultures treated as described in FIG. 7A.

FIG. 8A is a bar graph showing infectious HSV yield for cell culturesinfected with an acyclovir resistant mutant HSV, according to an exampleembodiment of the present invention.

FIG. 8B is a bar graph showing infectious HSV yield for cell culturesinfected with a different acyclovir resistant mutant HSV, according toan example embodiment of the present invention.

FIG. 9 is a bar graph showing replication of HSV-1 in primary cornealepithelial cell cultures treated with vehicle or peg-Arginase I,according to an example embodiment of the present invention.

FIG. 10 is line graph showing viral production in a rabbit eye model foreyes infected with HSV-1 and treated with controls (peg-BSA and PBS), 1%triflourothymidine, or peg-Arginase I, according to an exampleembodiment of the present invention.

FIG. 11 is a series of images of rabbit eyes infected with HSV-1 andtreated with controls (peg-BSA or PBS), 1% triflourothymidine, orpeg-Arginase I, according to an example embodiment of the presentinvention.

FIG. 12 is a line graph showing slit lamp examination scores over an 8day period for the eyes shown in FIG. 11.

FIG. 13 is a line graph showing combined ocular clinical scores over an8 day period for the eyes shown in FIG. 11.

FIG. 14 is a line graph showing corneal stromal inflammation over an 8day period for the eyes shown in FIG. 11.

FIG. 15 is a line graph showing scleral inflammation over an 8 dayperiod for the eyes shown in FIG. 11.

FIG. 16 is a line graph showing corneal neovascularization over an 8 dayperiod for the eyes shown in FIG. 11.

FIG. 17 is a line graph showing corneal eyelid inflammation over an 8day period for the eyes shown in FIG. 11.

FIG. 18 is a pair of images of cell cultures infected with Adenovirusand treated with control (peg-BSA) or peg-Arginase I drops, according toan example embodiment of the present invention.

FIG. 19 is a series of images of rabbit eyes with an Adenoviralinfection and treated with control (peg-BSA), cidofovir, or peg-ArginaseI drops, according to an example embodiment of the present invention.

FIG. 20 is a line graph showing slit lamp examination scores over an 8day period for the eyes shown in FIG. 19.

FIG. 21 is a line graph showing combined ocular clinical scores over an8 day period for the eyes shown in FIG. 19.

FIG. 22 is a line graph showing corneal neovascularization over an 8 dayperiod for the eyes shown in FIG. 19.

FIG. 23 is a line graph showing scleral inflammation over an 8 dayperiod for the eyes shown in FIG. 19.

FIG. 24 is a line graph showing corneal stromal inflammation over an 8day period for the eyes shown in FIG. 19.

FIG. 25A is a series of images comparing effects of treatment witheither control (peg-BSA) or peg-Arginase I on endothelial cell migrationin a scratch wound healing model, according to an example embodiment ofthe present invention.

FIG. 25B is a bar graph showing a percentage of unhealed wound areaafter a twenty-four hour period for the wounds shown in FIG. 25A.

FIG. 26A is a pair of images of rabbit eyes having a VEGF pelletsurgically implanted into a corneal micropocket and treated with vehicleor peg-Arginase I, according to an example embodiment of the presentinvention.

FIG. 26B is a bar graph showing areas of VEGF-mediated cornealneovascularization for the eyes shown in FIG. 26A

FIG. 27A is a series of images of a pair of rabbit eyes from the samerabbit showing clouding of the cornea in vehicle treated eyes, accordingto an example embodiment of the present invention.

FIG. 27B is a bar graph showing corneal cloudiness measurements for theeyes shown in FIG. 27A.

DESCRIPTION

Embodiments of the present disclosure provide a formulation of pegylatedarginase I (peg-Arginase I) that simultaneously treats inflammatoryreactions and neovascularization of corneal tissue, while promotinghealing of a damaged cornea. Embodiments may include peg-Arginase I aspart of an ophthalmic formulation, which may be applied topically to anaffected eye. Embodiments of the present disclosure provide methods forimproved treatment of ocular diseases whereby the methodssimultaneously: (1) inhibit damaging inflammatory reactions withoutblocking protective host responses; (2) inhibit replication of multiplepathogens; (3) prevent neovascularization processes; and (4) promotehealing. Further, embodiments of the present disclosure target pathwaysthat are unlikely to lead to the evolution of drug resistance and may beeffective against current drug resistant pathogens.

Embodiments of the present disclosure include methods of modulatingamino acid concentrations in microenvironments. Embodiments of thepresent disclosure modulate amino acid concentrations as a therapeuticapproach for viral infections. In certain embodiments, amino acidconcentrations are modulated to inhibit viral replication and preventdeleterious inflammation. In embodiments of the present invention,peg-Arginase I is administered as an antiviral. Embodiments of thepresent invention may be utilized to treat a broad range of viralinfections. In certain embodiments of the present disclosure,peg-Arginase I may be administered to a patient infected by a virus.Peg-Arginase I may be administered to deplete arginine in a recipient.Depletion of arginine may inhibit viral replication and/or inflammatoryresponses.

Arginase is a manganese-containing enzyme and is part of the urea cycle.In most mammals, two isozymes of arginase exist: (1) arginase I, whichfunctions in the urea cycle and is located primarily in the cytoplasm ofthe liver, and (2) arginase II, which may regulate arginine/ornithineconcentrations in the cell. Pegylation is the process of covalentattachment of polyethylene glycol (peg) polymer chains to anothermolecule such as a drug or protein. Pegylation may mask an agent from ahost's immune system and may provide increased solubility, mobility andlongevity to the agent. peg-Arginase I is a formulation of arginase Ithat has been pegylated.

In embodiments of the present invention, application of peg-Arginase Imay occur exogenously and may be delivered as a topical ophthalmicformulation such as an eye drop. This peg-Arginase I ophthalmic eye dropmay be composed of both protein conjugated and free peg molecules whichmay facilitate enzyme stability and mask the peg-Arginase I from hostresponses. Presence of free peg molecules may also impart properties tothe formulation that aid in peg-Arginase I's ophthalmic activities. Thepeg-Arginase I eye drop may contain various buffer systems that maintaina pH level that is conducive for Arginase enzymatic activity and istolerated by the eye. In certain embodiments, peg-Arginase I may beapplied in combination with other antivirals, anti-inflammatories oranti-inflammatory treatments. peg-Arginase I application is effective intreating corneal trauma, promoting healing of the cornea, and preventingdeleterious vision-threatening inflammation.

Embodiments of the present disclosure may be affective against both RNAand DNA viral infections, including both enveloped and non-envelopedviruses. Embodiments of the present disclosure may be used to treat avariety of viral infections, including but not limited to, ocularinfections and respiratory infections. Embodiments of the presentdisclosure may be administered orally or as an inhalant formulation.Embodiments of the present disclosure may be incorporated into aninjection composition.

Embodiments of the present disclosure may also be used to treat certainbacterial infections, including Chlamydia trachomatis. The presentdisclosure may be utilized to treat a variety of pathogen andinflammation-associated ocular diseases, including:Adenovirus-associated epidemic keratoconjunctivitis (no currenteffective treatment); herpes simplex-associated eye infections;varicella zoster-associated eye infections; herpes stromal keratitis;inflammation-associated diseases; CMV retinitis; and drug resistantpathogens. Moreover, embodiments may be used to treat humans or invarious veterinarian applications, including feline, canine, and equineocular diseases. The foregoing list is exemplary only and is notintended to identify all ocular diseases that may be treated with themethods identified herein.

In an exemplary embodiment, the present disclosure may be employed totreat ocular lesions, which may be caused by accidental or surgicaltrauma. Application of peg-Arginase I to a corneal lesion accelerateswound healing while reducing or eliminating ocular inflammation,neovascularization and inflammatory discharge. FIGS. 1 through 4 andTables 1 through 5 demonstrate experimental results of an embodiment ofthe present disclosure whereby surgically-induced corneal lesions weretreated with peg-Arginase I. FIG. 1A shows representative images of thelesions and demonstrates that treatment with an embodiment of thepresent invention accelerated healing of the lesion compared topeg-vehicle treatments. As shown in FIG. 1B and Tables 1 and 2, rates ofcorneal healing and injury closure were substantially faster whentreated with an embodiment of the present invention, with 100% of eyesreaching clinical cure for epithelial defects two days faster than thosetreated only with peg-vehicle.

TABLE 1 Calculated Times of Corneal Epithelium Closure % Closurepeg-ArgI Vehicle 25% 14 h 36 h 50% 30 h 48 h 75% 38 h 64 h

TABLE 2 Number of Eyes Clinically Cured for Area of Corneal DebridementNumber Eyes Number Eyes Clinically Cured Clinically Cured DAY (peg-ArgI)(Vehicle) 0 0/5 0/5 1 0/5 0/5 2 0/5 0/5 3 3/5 (60%) 1/5 (20%) 4 5/5(100%) 2/5 (40%) 6 5/5 (100%) 5/5 (100%)

Moreover, as shown in FIG. 2 and Table 3, peg-Arginase I treated eyesdid not experience an increase in presentation of any clinical symptoms,and combined clinical presentations markedly declined with treatment. Onday four, 80% of eyes treated with an embodiment of the presentinvention exhibited no symptomology from the surgically-induced traumawith all eyes clinically cured by day six. In contrast, only 40% ofvehicle treated eyes showed clinical cure for all ocular parameters byday six of treatment.

TABLE 3 Number of Eyes Clinically Cured of All Combined Ocular ClinicalConditions Number Eyes Number Eyes Clinically Cured Clinically Cured DAY(peg-ArgI) (Vehicle) 0 0/5 0/5 1 0/5 0/5 2 0/5 0/5 3 0/5 0/5 4 4/5 (80%)0/5 6 5/5 (100%) 2/5 (40%)

FIGS. 3 and 4, as well as Tables 4 and 5, illustrate that treatment withan embodiment of the present invention prevents cornealneovascularization and inflammation of the sclera in this corneal woundhealing model. These results not only demonstrate effectiveness of thepresent invention for treating traumatic ocular issues, but exemplifythat embodiments of the ophthalmic formulation are well-tolerated in theeye even in the presence of traumatic damage.

TABLE 4 Number of Eyes Clinically Cured of Corneal NeovascularizationNumber Eyes Number Eyes Clinically Cured Clinically Cured DAY (peg-ArgI)(Vehicle) 0 5/5 (100%) 5/5 (100%) 1 5/5 (100%) 0/5 2 5/5 (100%) 0/5 35/5 (100%) 0/5 4 5/5 (100%) 0/5 6 5/5 (100%) 3/5 (60%)

TABLE 5 Number of Eyes Clinically Cured of Scleral Inflammation(Injection) Number Eyes Number Eyes Clinically Cured Clinically CuredDAY (peg-ArgI) (Vehicle) 0 5/5 (100%) 5/5 (100%) 1 5/5 (100%) 1/5 (20%)2 5/5 (100%) 1/5 (20%) 3 5/5 (100%) 4/5 (80)% 4 5/5 (100%) 4/5 (80) 65/5 (100%) 5/5 (100%)

Accordingly, embodiments of the present invention are effective in thetreatment of ocular trauma, including reducing or eliminatinginflammation, neovascularization and/or inflammatory discharge.Embodiments may substantially accelerate healing of the eye after anocular trauma incident.

In another exemplary embodiment, the present disclosure may be employedto treat pathogenic infections of the eye (e.g., herpes simplex virustypes 1 and 2 (HSV-1 and HSV-2) and Adenovirus infections). Embodimentsmay be used as a treatment against wild-type and/or drug resistantherpes simplex virus (HSV) replication and/or transmission, as well asHSV-mediated inflammation-associated ocular disease. In FIG. 5, cellsinfected by HSV-1 and treated with an embodiment of the presentinvention inhibited production of infectious virus by greater than 1000fold even at high multiplicities of infection (MOIs). The presentinvention was at least as effective as high doses of a commonanti-herpetic, acyclovir. As shown in FIGS. 6 and 7, peg-Arginase Itreatment blocked virus-associated cytopathic effects, viraltransmission to uninfected cells, and cell-to-cell spread. FIGS. 8A and8B demonstrate that, for two different drug resistant mutant HSVviruses, peg-Arginase I is highly efficient at inhibiting drug-resistantviral replication, while anti-herpetic drugs, such as acyclovir, are notfunctional against such strains, even at high doses. FIG. 9 illustratesthat antiviral effects of peg-Arginase I were also observed in primaryhuman corneal epithelial cells, a cell line that recapitulates thenatural cells where herpes viruses replicate within the eye.

In an in vivo rabbit eye model system, the ability of peg-Arginase I toprevent HSV-mediated inflammation-associated disease was assessedrelative to a commonly prescribed ophthalmic antiviral-1%triflourothymidine (TFT). Rabbit eyes were infected with HSV, andinfection and disease were allowed to establish prior to beginningtreatment. FIG. 10 illustrates that at day three post infectionproduction of viral titers were matched, and that upon treatment withpeg-Arginase I, infectious viral titers were barely detectable. Theseresults were better than that observed for treatment with theanti-herpetic 1% TFT. FIG. 11 depicts representative overall clinicalpresentation of ocular herpetic disease. In either of the control (PBSor peg-BSA) treatment groups, severe blinding disease was observedfollowing infection, with presence of severe inflammatory discharge andblinding epithelial defects observed by slit lamp examination. Incontrast, both peg-Arginase I and 1% TFT showed marked reduction in thepresence of dendritic or geographic ulceration, and improved clinicalappearance. The statistical significance of these clinical scorescomparing between treatment groups is shown in Table 6 and indicatesthat five days of treatment with peg-Arginase I statistically improvedclinical presentation of disease and resolved many inflammation andneovascularization associated clinical parameters.

TABLE 6 Significance of Clinical Scores Between Treatment Groups (Pvalues; P > 0.05 = NS) Group Slit Lamp Combined Corneal Scleral StromalCorneal Blepharitis Comparison Examination Clinical Neovasc. Inflam.Infla. Epithelium (Eyelid Infl.) Peg-Argl vs peg-BSA Start of Tx 1.00001.0000 1.0000 1.0000 1.0000 1.0000 0.9395 Day 7 <0.0001 0.0032 <0.0001<0.0001 0.0002 0.0003 0.0163 Peg-Argl vs PBS Start of Tx 1.0000 1.00001.0000 1.0000 1.0000 1.0000 1.0000 Day 7 <0.0001 0.0088 0.0006 0.00490.0041 0.0426 0.0011 1% TFT vs PEG-BSA Start of Tx 1.0000 1.0000 1.00001.0000 1.0000 1.0000 0.9395 Day 7 <0.0001 NS 0.0129 0.0012 0.0041 0.0125NS 1% TFT vs PBS Start of Tx 1.0000 1.0000 0.9981 0.9999 1.0000 1.00001.0000 Day 7 <0.0001 NS NS NS 0.0355 NS 0.0163FIGS. 12 through 17 graphically depict assessment of the averages ofthese clinical parameters for each day throughout the duration of theexperiment. Taken together, these results indicate that peg-Arginase Itreatment not only prevents ocular replication of HSV, but can suppressand resolve virus-induced inflammation-associated disease and thwartvision-threatening vascularization of the cornea.

In another exemplary embodiment, the present disclosure may be employedto treat other inflammation-associated ocular diseases, such as thoseinduced by Adenovirus infection. Clinical presentation followingAdenoviral infection of the eye may proceed even when antiviralcompounds, such as cidofovir, are present. Clinical presentation ofAdenovirus-associated ocular disease may be due to uncontrolledinflammatory responses to the infection even following viral clearance.FIG. 18 illustrates that peg-Arginase I prevents Adenovirus-mediatedcellular cytopathic effects and exhibits no toxicity to cells.

In an in vivo rabbit eye model system, the ability of peg-Arginase I toprevent inflammation-associated disease following Adenoviral infectionwas examined. Rabbit eyes were infected with Adenovirus and eight hourslater treatment was initiated. Eyes were treated four times daily with10 U peg-Arginase I per 50 μl dose. FIG. 19 depicts representativeimages from the Adenovirus Type 5 “pink-eye” clinical disease rabbit eyemodel showing that peg-Arginase I treated eyes showed fewer clinicalsymptoms, including less corneal vascularization, epiphora, blepharitis,and scleral injection, compared to eyes treated with peg-BSA control orthe potent antiviral cidofovir. FIGS. 20 through 24 illustrate thatpeg-Arginase I suppresses clinical presentation of disease at alltreatment days, including suppression of ocular inflammation andneovascularization. These effects were independent of its ability toinhibit Adenoviral replication, since the antiviral cidofovir did notprevent clinical disease presentation. These properties of peg-ArginaseI illustrate the present invention's utility in preventing numerousvision-threatening ocular diseases that present clinically asinflammation or neovascularization of the eye.

In another exemplary embodiment, the present disclosure may be employedto prevent and resolve corneal neovascularization andvascularization-associated edema. As observed in all other modelsystems, peg-Arginase I treatment prevented and/or reduced presentationof vascularization of the cornea, a vision-threatening condition that isassociated with many ocular diseases. A key mediator and initiator ofcorneal vascularization may be vascular endothelial growth factor(VEGF). FIGS. 25A and 25B illustrate that peg-Arginase I preventsVEGF-mediated migration of primary vascular endothelial cells, aprerequisite for blood vessel growth and expansion onto the cornea.

In an in vivo rabbit eye model system, the ability of peg-Arginase I toprevent VEGF-mediated corneal vascularization was assessed by implantingslow release VEGF pellets within a corneal micropocket 3 mm from thecorneal limbus. Eyes were subsequently treated with either peg-ArginaseI (right, OD eye) or peg-BSA vehicle control (left, OS eye) 4 timesdaily at a dose of 10 U per 50 μl drop. FIGS. 26A and 26B demonstratethat the extent and areas of neovascularization of the cornea weresignificantly reduced in peg-Arginase I treated eyes compared to peg-BSAtreated controls. Moreover, even when vascularization was present, FIGS.27A and 27B establish that peg-Arginase I treatment prevented theappearance of corneal edema that resulted in corneal clouding andepithelial defects. As shown, peg-Arginase I inhibits vision-threateningcorneal vascularization, further enabling peg-Arginase I to prevent andameliorate vision-threatening aspects of many ocular diseases. Moreover,inhibition of vascularization assists in suppressing deleteriousinflammatory responses.

Although certain embodiments discussed herein address ocular challengemodels, embodiments of the present disclosure inhibit viral replication,neovascularization, and inflammatory responses without limitation to thelocation of the viral infection. Embodiments of the present disclosuremay be utilized to treat a variety of viral infections regardless ofwhether the infection is within a patient's eyes.

While the embodiments are described with reference to variousimplementations and exploitations, it will be understood that theseembodiments are illustrative and that the scope of the inventions is notlimited to them. Many variations, modifications, additions, andimprovements are possible. Further still, any steps described herein maybe carried out in any desired order, and any desired steps may be addedor deleted. The specification and drawings are accordingly to beregarded in an illustrative rather than in a restrictive sense.

What is claimed is:
 1. An anti-viral treatment, comprising:administering peg-Arginase I to a patient infected with a virus, whereinthe patient is infected by at least one of Herpes simplex virus-1(HSV-1), Herpes simplex virus-2 (HSV-2), Cytomegalovirus (CMV),Varicella zoster virus (VZV), Adenovirus, and Influenza virus, andwherein the at least one of HSV-1, HSV-2, CMV, VZV, Adenovirus, andInfluenza virus is drug resistant.